Reproduction (2001) 121, 677–683 Review

Mechanisms for pattern formation leading to axis formation and lineage allocation in mammals: a marsupial perspective

Lynne Selwood

Department of Zoology, University of Melbourne, Royal Parade, 3010 Vic, Australia

Developing patterns in early embryogenesis are analysed in conceptuses from several families, including Dasyuridae, Phalangeridae, Macropodidae and Didelphidae, in which has been examined in some detail. Features common to cleavage and formation, and in some cases to formation, are used to develop an outline of possible mechanisms leading to axis formation and lineage allocation. Relevant features that have been described only in some species are also included. It is suggested that certain features of marsupial cleavage establish patterns in the developing blastocyst epithelia, pluriblast, and hypoblast that contribute to axis formation and lineage allocation. All marsupials examined had a polarized oocyte or conceptus, the polarity of which was related to the conceptus embryonic–abembryonic axis and, eventually, the conceptus dorsal–ventral axis and the formation of the pluriblast (future ) and trophoblast. The embryonic dorsal–ventral and anterior–posterior axes were established after the allocation of hypoblast and . Mechanisms that appear to result in patterning of the developing epithelia leading to axis formation and lineage allocation are discussed, and include sperm entry point, gravity, conceptus polarity, differentials in cell–zona, cell–cell and cell-type (boundary effects) contacts, cell division order during cleavage and signals external to the conceptus. A model of the patterning effects is included. The applicability of these mechanisms to other amniotes, including eutherian mammals, is also examined.

Because of the simplified nature of blastocyst formation, environment by development of a placenta. This review will study of marsupial development has the potential to give show how a study of early marsupial development reveals insights into developmental mechanisms and evolution of potential mechanisms for lineage allocation and axis the mammalian embryo, especially with respect to axis formation. formation and lineage allocation. Marsupials do not form a but instead the undergo a mesenchyme– Conceptus and embryonic axes epithelial transformation during cleavage to form a unilaminar epithelium in which both the pluriblast (equiv- In amniotes, because early processes are concerned with alent to the ; ICM) and the trophoblast are establishing the extra-embryonic lineages and the embryo superficial in position. The terminology of Johnson and proper does not emerge until later, it is important to distinguish Selwood (1996), which allows equivalent structures in between axes developing with respect to the conceptus and different mammalian groups to be compared, is used in this those developing with respect to the embryo proper. The two review. axes are related but are not exactly the same (Fig. 1). Axis Marsupial studies may indicate some evolutionary formation and lineage allocation are intimately related in all processes because marsupial conceptuses have features in amniotes in which the separation of embryonic from extra- common with other amniotes. Like the conceptus of reptiles embryonic lineages is preceded by the establishment of the and birds the marsupial conceptus is covered by a number conceptus embryonic–abembryonic axis (E/AbE). The first of egg coats. The mode of formation of the early embryonic extra-embryonic lineages, trophoblast or extra-embryonic and extra-embryonic lineages is remarkably similar to that of the and then hypoblast (Fig. 1), have found in reptiles and birds, but can be readily seen because fundamental roles in conceptus nourishment and embryonic of the paucity of yolk material. Like the conceptus of signalling. They also are manifestations of the first signs of the eutherian mammals, the marsupial conceptus has to emerging dorsal–ventral (D–V) axis for the conceptus as a prepare itself to obtain nourishment from the uterine whole (Fig. 1). Within the epiblast, which gives rise to the future embryo (Johnson and Selwood, 1996), the definitive Email: [email protected] embryonic D–V and anterior–posterior (A–P) axes of the future

© 2001 Journals of Reproduction and Fertility 1470-1626/2001 Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access 678 L. Selwood

Table 1. Possible mechanisms leading to axial gradients or lineage allocation in marsupials

Mechanisms Evidence References

Uneven distribution of determinants Oocyte or polarity Selwood and Hickford, 1999 (review) Gravity Gravity-related orientation of conceptuses Baggott and Moore, 1990 in one species Sperm entry point Identified in some species showing marked Merry et al., 1995; Selwood, unpublished oocyte polarity before fertilization Cleavage patterns Stylized and specific morphology Selwood, 1992 Intrinsic signals Cell division order creates pattern in Selwood and Smith, 1990 pluriblast and trophoblast Extrinsic signals Developmental arrests in vitro Selwood, 1992 (review) organism emerges (Fig. 1). Apart from the small size, the nants. Direct determinants are defined as determinants the appearance of the marsupial epiblast during is presence of which results directly in axial gradients or similar to that of birds, reptiles and monotremes. lineage allocation. Indirect or pattern-forming determinants are determinants the presence of which creates a pattern leading eventually to axial gradients or lineage allocation. Possible mechanisms leading to axial gradients or Polarity may be related to the uneven distribution of direct lineage allocation determinants because the conceptus E/AbE, D–V axis and It is proposed here that axial gradients are set up during the first lineage allocation into pluriblast and trophoblast early development before the axis can be detected mor- appears to be related to the cytoplasmic polarity of the phologically. The processes in marsupials that have the zygote (Fig. 1). The fact that the differences between potential to create patterns in the developing blastocyst pluriblast and trophoblast often disappear soon after the epithelium leading to either axial gradients or lineage blastocyst epithelium is complete (one or two cell divisions) allocation, or both, are discussed below (see Table 1). does not support this contention and indicates that polarity is more likely to be related to the distribution of indirect Conceptus axis formation and pluriblast–trophoblast determinants to ensure that formation of the blastocyst allocation epithelium is localized to one hemisphere. These two alternatives need to be tested experimentally. After cell– Oocyte–zygote polarity. Oocyte–zygote polarity is a zona adhesion begins (Fig. 2), polarity in trophoblast and feature encountered commonly in most marsupial families pluriblast cells becomes related to the blastocyst epithelium, (Figs 1 and 2) and is expressed in the eccentric location of the outer surface of which is apical (Frankenberg and the nucleus or other organelles in oocytes and cleavage Selwood, 1998). This process is in contrast to that in stage conceptuses, the polarized discharge of pale vesicles eutherian mammals, in which only trophoblast cells are releasing an extracellular matrix (ECM) into the cleavage polarized. cavity or vesicles associated with cell–zona adhesion, the polarized nature of cell–zona adhesion, which is always Sperm entry point. Sperm entry point appears to occur localized initially to the hemisphere opposite vesicle preferentially in the hemisphere opposite the accumulation emission (Fig. 3b) and the asymmetric mucoid coat in some of vesicles that contribute to the yolk mass in dasyurid species (Selwood and Hickford, 1999). All early concep- marsupials (Table 1) and within 60Њ of the position of the tuses show polarity but do not all share the same polarized first polar body (Fig. 3). This preference may occur because features. However, all early conceptuses that have been penetration in the hemisphere containing the vesicular analysed ultrastructurally show polarity related to the material of the future yolk mass makes migration of the distribution of vesicles at one pole and nucleus or sperm head difficult or impossible. If the relationship mitochondrial-rich cytoplasm at the other (Selwood and between oocyte polarity, sperm entry point and the future Sathananthan, 1988; Baggott and Moore, 1990; Renfree D–V axis is examined in dasyurids, some parallels with the and Lewis, 1996; Frankenberg and Selwood, 1998). A relationship in the frog can be seen (Fig. 3) that indicate that variety of anchoring devices characterize marsupial the sperm entry point may be associated with formation of so that the mitochondrial-rich cytoplasm is retained when the conceptus D–V axis. This hypothesis needs to be tested vesicles or the yolk mass are discharged (Breed et al., 1994, experimentally. If the sperm entry point is demonstrated to 1995; Merry et al., 1995; Frankenberg and Selwood, 1998). play a role in conceptus D–V axis formation, the move- Oocyte polarity may be related to polarized distribution ments of the cytoplasm after fertilization may be in response of either direct determinants or indirect maternal determi- to a gravitational stimulus (Table 1).

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access Pattern formation in the marsupial conceptus 679

(a)E (b) E (c) D*

*

AbE AbE V*

(d)D (e) (f)

A V V V DV

P

V*

Fig. 1. Schematic representation of the developing conceptus axes and their relationship to the embryonic axes in marsupials. (a–d) Diagrammatic transverse sections. (e–f) External view of dorsal surface. (a) Zygote with polarized location of the pronuclei (closed black ovals) in vesicle-free cytoplasm (dark blue oval) and vesicle-rich cytoplasm (yellow circles). The position of the conceptus embryonic–abembryonic (E/AbE) axis is shown. (b) Unilaminar blastocyst stage in which the pluriblast (pale brown) cells are distinct from the trophoblast cells (blue) and aligned along the E/AbE axis. Pluriblast cells are more rounded and larger than the more flattened, more vesicular trophoblast cells (not shown here). (c) Early bilaminar blastocyst stage in which the allocation of hypoblast (orange stars) from the pluriblast cells marks the transition to the conceptus dorso–ventral (D*/V*) axis. Hypoblast cells originate from the pluriblast by directed mitosis or by inward migration either from one side (*arrow) as in Antechinus stuartii or from the entire margin (arrow and *arrow) of the pluriblast as in Didelphis virginiana. (d) Trilaminar blastocyst stage in which developing mesenchymal (black) lineages between the hypoblast (orange) and overlying epiblast (yellow) initiate the transition to the embryonic D–V axis (dashed lines) which is restricted to the region of the epiblast. The D pole is common to both conceptus D–V* and embryonic D–V axis. The proximal and distal trophoblast also become distinct (not shown) aligned to the conceptus D–V* axis. (e) Dorsal surface view of a bilaminar blastocyst showing the circular epiblast with no overt signs of axial gradients. (f) Dorsal surface view of an early trilaminar blastocyst in which the anterior–posterior (A–P) axis is defined initially by the increased density of epiblast cells posteriorly (not shown), the oval shape of the epiblast, then the (black line), which extends posteriorly and also marks the most dorsal region of the embryonic D–V axis.

Cleavage patterns. The association of conceptus polarity Each pattern shows differences in the timing of features so and the stylized and consistent cleavage patterns specific to characteristic of marsupial cleavage. These characteristic a taxonomic group (Fig. 2) is the most powerful evidence for features are: the stage of overt polarization, polarized uneven distribution of determinants in marsupial zygotes. vesicle or yolk mass elimination, cell–zona adhesion, Cleavage patterns are holoblastic, showing accumulation of cell–cell adhesion, allocation of pluriblast and trophoblast ECM into the cleavage cavity during early cleavage (Fig. 2). and the formation of a complete unilaminar blastocyst. Only some patterns have polarized elimination of a large or several smaller membrane-bounded yolk masses and polar- Epiblast–hypoblast allocation and embryonic axis ized discharge of ECM (Figs 2 and 3b). During cleavage, the formation E/AbE axis, visible in most zygotes from the position of the nucleus (Fig. 2), becomes clearly established. The types of After establishment of the conceptus E/AbE and D–V axes cleavage pattern found in marsupials, in which cleavage and allocation of pluriblast–trophoblast lineages, the allo- has been described to the 32-cell stage are shown (Fig. 2). cation of epiblast, hypoblast, proximal and distal tro-

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access 680 L. Selwood

1 -C 2 -C 4 -C 8 -C 16 -C CÐZ∗ CÐC

D.v.

CÐZ CÐC ∗

T.v.

CÐZ CÐC ∗

S.m.

CÐZ CÐC

M.e.

Fig. 2. Diagrams of the cleavage pattern of four marsupials, Didelphis virginiana (D.v.), Trichosurus vulpecula (T.v.) and Sminthopsis macroura (S.m.), in which all of cleavage has been described, and Macropus eugenii (M.e.) in which cleavage has been described to the eight-cell stage. The pattern in T. vulpecula resembles that in Monodelphis domestica and that in S. macroura resembles that in Antechinus stuartii and Dasyurus viverrinus. Representative sections through five cleavage stages (one- cell to 16-cell) are shown, together with the stage at which cell–zona adhesion (C–Z), cell–cell adhesion (C–C) and formation of pluriblast and trophoblast (*) occur. Yolk masses: large closed yellow ovals and circles; pronuclei: closed black ovals; extracellular matrix: pale yellow circles in one-cell stages, and pale yellow colour in cleavage cavity in others; future pluriblast and undesignated cells: pale brown; and future trophoblast cells: blue.

phoblast and the emergence of the embryonic A–P and D–V result of the uneven distribution of indirect pattern-forming axes occur (Fig. 1). The most likely hypothesis in marsupials determinants or positional signals, or both. Positional is that these features emerge as a result of patterns occurring signals may be intrinsic to the embryo (that is, within the within the developing pluriblast and trophoblast epithelia. pluriblast or epiblast) or extrinsic (that is, within the This hypothesis would fit with the capacity to regulate trophoblast or hypoblast or external to the conceptus). An shown by the two most studied model amniote systems, underlying process regulating the pluriblast or trophoblast, those of the chick and the mouse. Capacity to regulate is an similar to the intercalation rule suggested for regenerating early feature and includes regulation for deletion or systems (Bryant et al., 1977), would have to apply to addition of cells to produce a normal blastocyst with the maintain the patterns leading to hypoblast allocation and ICM and trophoblast in mice (Tarkowski and Wroblewska, embryonic axis formation. The intercalation rule states 1967) and the rebuilding of a node in the epiblast in chicks that where a pattern in a field is disrupted, cells are (Yuan et al., 1995). intercalated to make up the cells with the missing values. Marsupials do not seem to be able to replace cells Unlike regenerating systems in secondary fields, destroyed in their developing blastocyst epithelia during intercalation in the pluriblast or epiblast may be achieved cleavage, but once the epithelia are established, damaged without proliferation. cells are quickly replaced (Selwood, 1986). The lineage potency of these cells has not been tested. It is the capacity Intrinsic signals to regulate that argues most strongly against the idea that lineage allocation or axis formation is dependent on uneven The pluriblast epithelium of marsupials, unlike the ICM distribution of direct determinants. This regulatory capacity of mice, is unilaminar, polarized and superficial in position. indicates that patterns form in the developing epithelia as a This means that differential signals related to an outside

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access Pattern formation in the marsupial conceptus 681

the potential sources of intrinsic signals causing variations (a) (b)E (c) D∗ to occur within the pluriblast epithelium of marsupials? Some indirect sources may be associated with uneven ∗ distribution of determinants, similar to those that occur in the vasa protein in the chick germ cell lineage (Tsunekawa et al., 2000). Several obvious candidates for intrinsic positional signals can be found during development of 60° 60° the epithelia, namely: differential in cell–zona contacts; AbE V∗ differential in cell–cell contacts; boundary effects, or (d) (e) differential in cell type contacts; and differential in order of cell division.

FOFF LL Differential in cell–zona contacts. Cells developing in the centre of the developing epithelium have earlier cell–zona contacts than those at the periphery. Furthermore, the nature and mechanisms of establishment of cell–zona contacts appears to vary between central and peripheral cells in two species, Antechinus stuartii (Sathananthan et al., 1997) and Fig. 3. Diagrams of the developing patterns in two dasyurid Trichosurus vulpecula (Frankenberg and Selwood, 1998). marsupials, Sminthopsis macroura and Antechinus stuarti. (a–c) This variation may be a mechanism to allocate pluriblast Transverse sections showing the relationship between sperm entry and trophoblast, or a consequence of the allocation. This point, conceptus polarity and the developing conceptus embryonic– mechanism may also separate central from peripheral cells abembryonic (E/AbE) and dorsal–ventral (D*–V*) axes. (d–e) Views within the pluriblast, and proximal from distal trophoblast from the dorsal side of the conceptus showing how the order of cell (Fig. 2). division can create an axis across the conceptus. (a) Polarized oocyte has a yolk mass (yellow oval at one pole with the first polar Differential in cell–cell contacts. In dasyurids, cells at the body (blue oval) and meiotic apparatus (diamond) at the other). centre of the developing epithelium have more and earlier Sperm entry point occurs within 60Њ of the polar body. (b) Four-cell stage, showing the conceptus E/AbE axis and the polarized nature cell–cell contacts (Selwood and Hickford, 1999). In of secretions (broad arrows) leading to cell–zona adhesion at the contrast, in the didephid Didelphys virginiana peripheral embryonic pole and polarized secretion of ECM (small arrows) into cells initially have more cell–cell contact (McCrady, 1938). the abembryonic hemisphere of the cleavage cavity (pale yellow). This differential would re-enforce the pattern established by (c) Early bilaminar blastocyst in which hypoblast cells (orange the differential in cell–zona contacts. stars) and the pluriblast (pale brown arc) define the conceptus dorsal (D*) pole and the trophoblast (blue semi-circle) defines the Boundary effect or differential in cell type contacts. Cells ventral pole (V*). Hypoblast cells first appear on one side of the within the inner pluriblast have pluriblast–pluriblast pluriblast margin (*). (d) Four-cell stage, in which cell division is contacts. Cells at the boundary of the pluriblast also have asynchronous, showing the four blastomeres lying above the yolk pluriblast–trophoblast contacts (Fig. 4a). Similar principles mass (yellow circle). The first cell to divide (F) and the cell opposite (O) it are marked. (e) The same embryo at the 16-cell stage, apply in the trophoblast. The establishment of a boundary showing how at the fifth division descendants (F) of the first cell to between the pluriblast and the trophoblast has important divide at the four-cell stage lie adjoining or nearby in the pluriblast consequences for future positional signalling between the (F in pale brown cell) and trophoblast (F in blue cell) are the first to tissues and probably contributes to the specification of divide in each lineage. The descendants of the cell opposite the ventral pluriblast–epiblast cells. With the formation of the first cell to divide at the four-cell stage lie on the other side of the hypoblast, additional boundary effects may be created conceptus and are the last cells to divide in each lineage of the between hypoblast and pluriblast or trophoblast (Fig. 4d). pluriblast (L in pale brown cell) and trophoblast (L in blue cell). Restriction of the first and last cells to divide to opposite sides of the Differential in order of cell division. In Sminthopsis conceptus has the potential to create an axis across it. macroura and Antechinus stuartii (Selwood and Smith, 1990), the descendants of the first cell to divide at the four- cell stage give rise to the first cell to divide at subsequent stages and lie on one side of the pluriblast and trophoblast (Fig. 3d,e). Descendants of the cell opposite it lie on the (that is, adjoining the external environment) versus an inside other side and are the last cells to divide at the eight-cell location (that is, adjoining the blastocoele) to stimulate stage onwards in both pluriblast and trophoblast. This development of the hypoblast or primitive mechanism may generate a hypoblast from one side of the (Gardner, 1982) do not occur in marsupials, in which the pluriblast and eventually an A–P axis. It is not necessarily unilaminar form means that all pluriblast cells have a a common mechanism because, in D. virginiana, the similar position with respect to inside and outside. What are descendants of the first cell to divide at the two-cell stage

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access 682 L. Selwood

Relatively standard media formulations, supplemented with (a) (b) fetal calf serum, support culture for much of marsupial development but critical periods of development involving new lineage specification do not occur in vitro, indicating that at these times developmental progress is dependent on some unknown extrinsic signal(s). These critical stages are the proliferation of pluriblast and trophoblast after their forma- tion, hypoblast formation and the early stages of embryonic axis formation (Selwood and Hickford, 1999). (c) (d) Conclusions Study of marsupial provides a unique opportunity to examine the processes of axis formation and lineage allocation in amniotes. It may be that the processes revealed by this study are unique to marsupials, but it is much more likely that at least some of the spectrum of mechanisms used by marsupials to generate new lineages and axes will also be found in other amniotes. Fig. 4. Proposed patterns in conceptus epithelia in marsupials. (a–c) Surface views of a blastocyst from the dorsal side showing the This work was supported by the Australian Research Council. pluriblast–epiblast (yellow) and trophoblast (pale blue). (a) Potential boundary effects patterning (transparent blue overlay) contributed by differentials in cell–zona and cell–cell contacts so References that cells at the centre of each of the pluriblast and trophoblast Key references are identified by asterisks. epithelia are different from cells in the boundaries. (b) Potential Baggott LM and Moore HDM (1990) Early of the pattern contributed by differentials in cell-division order (black grey short-tailed opossum, Monodelphis domestica, in vivo and in vitro. spots) so that one side of the conceptus is different from the other. Journal of Zoology (London) 222 623–639 (c) Combination of the potential patterns of (a) and (b). (d) Breed WG (1996) Egg maturation and fertilization in marsupials Transverse section of a bilaminar blastocyst showing the above Reproduction, Fertility and Development 8 617–643 patterns superimposed over the epiblast (yellow arc) and Breed WG, Simerly C, Navara CS, VandeBerg JL and Schatten G (1994) trophoblast (blue) and the hypoblast (orange circle). Microtubule configurations in oocytes, zygotes and early embryos of a marsupial, Monodelphis domestica. 164 230–240 Bryant PJ, Bryant SV and French V (1977) Biological regeneration and pattern formation Scientific American 237 66–81 Frankenberg S and Selwood L (1998) An ultrastructural study of the role of an give rise to the upper tier of cells (McCrady, 1938) that later extracellular matrix during normal cleavage in a marsupial, the brushtail give rise to the future pluriblast cells (Hartman, 1916). It possum Molecular Reproduction and Development 50 420–433 would be useful to see a re-evaluation of the cleavage *Gardner RL (1982) Investigation of cell lineage and differentiation in the pattern in D. virginiana using ultrastructural analysis. extraembryonic endoderm of the mouse embryo Journal of and Experimental Morphology 68 175–198 The combined effect of these intrinsic signalling *Hartman CG (1916) Studies in the development of the opossum Didelphys mechanisms has the potential to create the patterns (Fig. 4) virginiana I. History of the early cleavage II. Formation of the blastocyst that lead to axial gradients and lineage allocation in the Journal of Morphology 27 1–83 epiblast. These patterning mechanisms, which are more *Johnson MH and Selwood L (1996) Nomenclature of early development in readily detected in marsupials, might also apply to amniotes mammals Reproduction, Fertility and Development 8 759–764 McCrady E, Jr (1938) The embryology of the opossum American Anatomy in general, including eutherian mammals. Memoirs 16 1–233 Merry NE, Johnson MH, Gehring CA and Selwood L (1995) Cytoskeletal Extrinsic signals organization in the oocyte, zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura) Molecular Reproduction Once a pattern is formed and maintained (Fig. 4), differential and Development 41 212–224 gene expression across the epithelium can occur in response Renfree MB and Lewis AM (1996) Cleavage in vivo and in vitro in the marsupial to a variety of positional signals either intrinsic or extrinsic, or Macropus eugenii. Reproduction, Fertility and Development 8 725–742 both. A number of diverse studies indicate that marsupial Sathananthan AH, Selwood L, Douglas I and Nanayakkara K (1997) Early cleavage to formation of the unilaminar blastocyst in the marsupial embryos do respond to extrinsic signals preceding new Antechinus stuartii: ultrastructure Reproduction, Fertility and phases in lineage allocation and axis formation. The nature of Development 9 201–212 these signals is not understood. Extrinsic signals are thought Selwood L (1986) Cleavage in vitro following destruction of some to initiate and terminate embryonic diapause and other blastomeres in the marsupial Antechinus stuartii (Macleay) Journal of Embryology and Experimental Morphology 92 71–84 developmental arrests in marsupials (Tyndale-Biscoe and *Selwood L (1992) Mechanisms underlying the development of pattern in Renfree, 1987). Hypoblast formation and the development of marsupial embryos. In Current Topics in Developmental Biology Vol. 27 the A–P axis follow the termination of embryonic diapause. Ed. RA Pedersen pp 175–233. Academic Press, New York

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access Pattern formation in the marsupial conceptus 683

Selwood L and Hickford D (1999) Early cell lineages in marsupial embryos. mouse eggs isolated at the 4- and 8-cell stage Journal of Embryology and In Cell Lineage and Fate Determination pp 505–519 Ed. SA Moody. Experimental Morphology 18 155–180 Academic Press, San Diego, CA Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T (2000) Isolation of Selwood L and Sathananthan AH (1988) Ultrastructure of early cleavage chicken vasa homolog gene and tracing the origin of primordial germ and yolk extrusion in the marsupial Antechinus stuartii. Journal of cells Development 127 2741–50 Morphology 195 327–344 Tyndale-Biscoe CH and Renfree MB (1987) Reproductive Physiology of Selwood L and Smith D (1990) Time-lapse analysis and normal stages of Marsupials Cambridge University Press, Cambridge development of cleavage and blastocyst formation in the marsupials the Yuan S, Darnell DK and Schoenwolf GC (1995) Mesodermal patterning brown antechinus and the stripe-faced dunnart Molecular Reproduction during avian gastrulation and : experimental induction of and Development 26 53–62 notochord from non-notochordal precursor cells Developmental *Tarkowski A and Wroblewska J (1967) Development of blastomeres of Genetics 17 38–54

Downloaded from Bioscientifica.com at 09/28/2021 08:12:18PM via free access